PURIFICATION AND CHARACTERIZATION OF A NOVEL SERINE PROTEINASE FROM THE MICROSOMAL FRACTION OF BOVINE PANCREAS

A novel, trypsin-like serine proteinase was purified to homogeneity fr om the bovine pancreas microsome fraction. The enzyme was solubilized with lamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), and p urified by a series of column chromatographic steps on Ultrogel AcA-34 , trypsin inhibitor-Sepharose 4B, and arginine-Sepharose 4B. The molec ular mass of this pancreas trypsin-like proteinase (bPTLP) was estimat ed to be 29.5 kDa by SDS-PAGE under reducing conditions. The NH2-termi nal sequence of bPTLP is very homologous, but not identical to those o f other serine proteinases, especially such as elastases IV, II, and I II, Substrate specificity studies involving a synthetic substrate and glucagon indicated that the enzyme hydrolyzes Arg-X, Lys-X, and Leu-X bonds. The best synthetic substrate for bPTLP was t-butyloxycarbonyl G ln-Arg-Arg-4-methylcoumaryl 7-amide. The enzyme failed to hydrolyze th e substrate for chymotrypsin and elastase. The enzyme activity was inh ibited by diisopropyl fluorophosphate, p-amidinophenylmethane sulfonyl fluoride, and leupeptin, indicating that it is a serine-proteinase. Th ese findings show that bPTLP is a novel serine-proteinase which differ s from all known proteinases. The physiological function of the enzyme has yet to be determined.